Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of blastocyst-stage embryos. Their importance to modern biology and regenerative medicine derives from two unique characteristics that distinguish them from all other organ-specific stem cells identified so far. First, hESCs can be maintained and expanded as pure population of undifferentiated cells for extended periods of time in culture. Second, hESCs can differentiate into every cell type in the body, including neuronal, cardiac, hepatic, and endothelial cells. However, one of the most devastating risks of using hESCs remains the possibility of cellular misbehavior (i.e., teratoma formation). Thus, understanding the biological process of hESC differentiation in vitro and in vivo is a must if the clinical potential of these cells is to be realized. This R21 proposal is a systemic approach to study this problem, which is an under-addressed issue in stem cell research at present. In Aim 1, we will monitor the dynamics of hESC misbehavior in living animals. In Aim 2, we will investigate the differentiation process of hESCs into endothelial cells. Collectively, we believe data gathered from this proposal will provide vital information needed for safe clinical translation of hESC-based therapy in the future. PROJECT NARRATIVE: Human embryonic stem cells (hESCs) have the unique ability to undergo both self-renewal and multi- lineage differentiation. This opens up exciting opportunities for understanding embryonic development on a basic research level and for application of hESC-based regenerative medicine on a clinical level. However, the fundamental biology of how these cells behave or misbehave in vivo still remains unknown. In particular, understanding how hESCs differentiate into specific cell lineages versus others will be critically important for harnessing their potential clinical values. In this proposal, we will integrate our expertise in molecular imaging, genomics, and proteomics to answer these questions using two federally approved hESC lines (H1 and H9).